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Concentration–response evaluation of ToxCast compounds for multivariate activity patterns of neural network function

Abstract

The US Environmental Protection Agency’s ToxCast program has generated toxicity data for thousands of chemicals but does not adequately assess potential neurotoxicity. Networks of neurons grown on microelectrode arrays (MEAs) offer an efficient approach to screen compounds for neuroactivity and distinguish between compound effects on firing, bursting, and connectivity patterns. Previously, single concentrations of the ToxCast Phase II library were screened for effects on mean firing rate (MFR) in rat primary cortical networks. Here, we expand this approach by retesting 384 of those compounds (including 222 active in the previous screen) in concentration–response across 43 network activity parameters to evaluate neural network function. Using hierarchical clustering and machine learning methods on the full suite of chemical-parameter response data, we identified 15 network activity parameters crucial in characterizing activity of 237 compounds that were response actives (“hits”). Recognized neurotoxic compounds in this network function assay were often more potent compared to other ToxCast assays. Of these chemical-parameter responses, we identified three k-means clusters of chemical-parameter activity (i.e., multivariate MEA response patterns). Next, we evaluated the MEA clusters for enrichment of chemical features using a subset of ToxPrint chemotypes, revealing chemical structural features that distinguished the MEA clusters. Finally, we assessed distribution of neurotoxicants with known pharmacology within the clusters and found that compounds segregated differentially. Collectively, these results demonstrate that multivariate MEA activity patterns can efficiently screen for diverse chemical activities relevant to neurotoxicity, and that response patterns may have predictive value related to chemical structural features.

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Acknowledgements

The authors acknowledge the outstanding tissue culture and general laboratory support of Ms Theresa Freudenrich at the US Environmental Protection Agency (EPA). In addition, the authors thank Drs Katie Paul-Friedman and Holly Mortensen at the US EPA for their useful comments on a previous version of this manuscript.

Funding

This work was supported in part by the National Health and Environmental Effects Research Laboratory (NHEERL) and in part by CRADA 644-11 between the US EPA and Axion Biosystems. This work was supported by the National Institutes of Health [ES025128, ES030007] and the US Environmental Protection Agency [STAR R835802].

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Correspondence to Timothy J. Shafer.

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Conflict of interest

JDS was an employee of Axion Biosystems when the data collection for this work was conducted. Axion is a manufacturer of microelectrode array recording equipment and supplies. All other authors declare that they have no conflicts of interest.

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Marissa B. Kosnik and Jenna D. Strickland contributed equally to this manuscript.

This document has been subjected to review by US EPA Office of Research and Development and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. This work was supported in part by CRADA 644–11 between the US EPA and Axion Biosystems.

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Supplementary file1 (XLSX 1080 kb)

204_2019_2636_MOESM2_ESM.pdf

Figure S1. Dendrogram from complete hierarchical clustering with Euclidian distance function adjusted to include differences between chemical-parameter AC50s and winning AIC models. Left= All parameters, Right= Reduced parameter set after removal of redundant parameters (PDF 332 kb)

204_2019_2636_MOESM3_ESM.pdf

Figure S2. Comparison of ToxCast assay potencies to most potent MEA parameter for each compound. Triangle= the log10(AC50) for the most potent MEA parameter for each chemical, Red= known neuroactive compounds (Table S1), Blue= other compounds (PDF 30 kb)

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Kosnik, M.B., Strickland, J.D., Marvel, S.W. et al. Concentration–response evaluation of ToxCast compounds for multivariate activity patterns of neural network function. Arch Toxicol 94, 469–484 (2020). https://doi.org/10.1007/s00204-019-02636-x

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  • DOI: https://doi.org/10.1007/s00204-019-02636-x

Keywords

  • Neurotoxicity
  • Screening
  • ToxCast